A Substituted Tetrahydroisoquinoiline Derivative as a D1 Positive Allosteric Modulator

ABSTRACT

The present invention relates to compound according to formula (I), (I) which is a positive allosteric modulator of D1 and accordingly of benefit as pharmaceutical agent for the treatment of diseases in which D1 receptors play a role.

The invention relates to a tetrahydroisoquinoline derivative and its use in therapy. In particular the present invention relates to a pharmacologically active substituted tetrahydroisoquinoline derivative.

This compound acts as a D1 Positive Allosteric Modulator and is accordingly of benefit as a pharmaceutical agent for the treatment of diseases in which D1 receptors play a role.

The monoamine dopamine acts via two families of GPCRs to modulate motor function, reward mechanisms, cognitive processes and other physiological functions. Specifically, dopamine is acting upon neurons via D1-like, comprising dopamine D1 and D5, receptors which couple mainly to the Gs G-protein and thereby stimulate cAMP production, and D2-like, which comprise D2, D3 and D4, receptors which couple to Gi/qG-proteins and which attenuate cAMP production. These receptors are widely expressed in different brain regions. In particular, D1 receptors are involved in numerous physiological functions and behavioural processes. D1 receptors are, for instance, involved in synaptic plasticity, cognitive function and goal-directed motor functions, but also in reward processes. Due to their role in several physiological/neurological processes, D1 receptors have been implicated in a variety of disorders including cognitive and negative symptoms in schizophrenia, cognitive impairment related to neuroleptic therapy, Mild Cognitive Impairment (MCI), impulsivity, Attention-Deficit Hyperactivity Disorder (ADHD), Parkinson's disease and other movement disorders, dystonia, Parkinson's dementia, Huntington's disease, dementia with Lewy Body, Alzheimer's disease, drug addiction sleep disorders, apathy, traumatical spinal cord injury or neuropathic pain.

It has proven difficult to develop orally-bioavailable small molecules targeting D1 receptors. D1 agonists developed so far are generally characterized by a catechol moiety and their clinical use has therefore been limited to invasive therapies. Achieving sufficient selectivity has also been challenging due to the high degree of homology in the ligand binding site between dopamine receptors subtypes (e.g. dopamine D1 and D5). Also, D1 agonists are associated with potentially limiting side effects including but not limited to dyskinesia and hypotension.

There is therefore a need to design new agents that could modulate D1 receptors.

There has been much interest in the identification of allosteric modulators of GPCRs, both as tools to understand receptor mechanisms and as potential therapeutic agents. GPCRs represent the largest family of cell-surface receptors and a large number of marketed drugs directly activate or block signaling pathways mediated by these receptors. However, for some GPCRs (e.g. peptide receptors), it has proven challenging to develop small molecules or to achieve sufficient selectivity due to the high degree of homology in the ligand binding site between subtypes (e.g. dopamine D1 and D5 or D2 and D3). Accordingly, much drug research has shifted to the identification of small molecules which target sites distinct from the orthosteric natural agonist. Ligands which bind to these sites induce a conformational change in the GPCR thereby allosterically modulating the receptor function. Allosteric ligands have a diverse range of activities including the ability to potentiate (positive allosteric modulator, PAM) or attenuate (negative allosteric modulator, NAM) the effects of the endogenous ligand, by affecting affinity and/or efficacy. As well as subtype selectivity, allosteric modulators may present other potential advantages from a drug discovery perspective such as a lack of direct effect or intrinsic efficacy; only potentiating the effect of the native transmitter where and when it is released; reduced propensity for inducing desensitization arising from constant exposure to an agonist as well as reduced propensity to induce target-related side-effects.

The compounds according to the present invention potentiates the effect of D1 agonists or of the endogenous ligand on D1 receptors through an allosteric mechanism, and is therefore a D1 Positive Allosteric Modulator (D1 PAM).

The compound in accordance with the present invention, being a D1 PAM, is therefore beneficial in the treatment and/or prevention of diseases and disorders in which D1 receptors play a role. Such diseases include cognitive and negative symptoms in schizophrenia, cognitive impairment related to neuroleptic therapy, Mild cognitive impairment (MCI), impulsivity, Attention-Deficit Hyperactivity Disorder (ADHD), Parkinson's disease and other movement disorders, dystonia, Parkinson's dementia, Huntington's disease, dementia with Lewy Body, Alzheimer's disease, drug addiction, sleep disorders, apathy, traumatic spinal cord injury or neuropathic pain.

International patent application WO 2013/051869 A1 discloses certain 3,4-dihydro-1H-isoquinolin-2-yl derivatives which are NK2 antagonists.

International patent application WO2008/109336 A1 discloses certain tetrahydroisoquinoline compounds which are modulators of the histamine H3 receptors.

International patent application WO2014/193781 A1 discloses certain 3,4-dihydroisoquinolin-2(1H)-yl derivatives useful for the treatment of cognitive impairment associated with Parkinson's disease or Schizophrenia.

International patent application WO2016/055479 discloses substituted 3,4-dihydroisoquinolin-2(1H)-yl derivatives and analogs thereof which may be useful for the treatment of diseases in which D1 receptors play a role.

International patent application WO2019/204418 discloses certain pyrazo-tetrahydroisoquinolines derivatives which are D1 positive allosteric modulators and may be useful in the treatment of Parkinson's disease, Alzheimer's disease, schizophrenia, and Attention-Deficit Hyperactivity Disorder (ADHD).

However, there remains a need to develop potent D1 positive allosteric modulators combining advantageous pharmacokinetic and pharmacodynamic properties while reducing side effects traditionally associated with treatments involving selective D1 agonists, such as for example hypotension or dyskinesia.

The present invention provides 2-(3,5-dichloro-1-methyl-indazol-4-yl)-1-[(1S,3R)-3-(hydroxymethyl)-5-(1-hydroxy-1-methyl-ethyl)-1-methyl-3,4-dihydro-1H-isoquinol in-2-yl]ethenone of formula (I),

-   -   or a pharmaceutically acceptable salt thereof.

The present invention also provides a compound of formula (I) as defined above or a pharmaceutically acceptable salt thereof, for use in therapy.

In another aspect, the present invention also provides a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for use in the treatment of diseases and/or disorders in which D1 receptors play a role.

In another aspect, the present invention provides a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of cognitive and negative symptoms in schizophrenia, cognitive impairment related to neuroleptic therapy, Mild Cognitive impairment (MCI), impulsivity, Attention-Deficit Hyperactivity Disorder (ADHD), Parkinson's disease and other movement disorders, dystonia, Parkinson's dementia, Huntington's disease, dementia with Lewy Body, Alzheimer's disease drug addiction, sleep disorders, apathy, traumatic spinal cord injury or neuropathic pain.

In a particular embodiment of this aspect, the present invention provides a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof for use in the treatment of Parkinson's disease and other movement disorders, Alzheimer's disease, or cognitive and negative symptoms in schizophrenia.

Therefore, in one particular aspect, the present invention provides a compound of formula (I), as defined above, or a pharmaceutically acceptable salt thereof, for use in the treatment of Parkinson's disease and other movement disorders.

In a further aspect, the present invention provides for the use of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament useful for the treatment and/or prevention of diseases and/or disorders in which D1 receptors play a role.

In another further aspect, the present invention provides for the use of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament useful for the treatment and/or prevention of cognitive and negative symptoms in schizophrenia, cognitive impairment related to neuroleptic therapy, Mild Cognitive Impairment (MCI), impulsivity, Attention-Deficit Hyperactivity Disorder (ADHD), Parkinson's disease and other movement disorders, dystonia, Parkinson's dementia, Huntington's disease, dementia with Lewy Body, Alzheimer's disease, drug addiction, sleep disorders, apathy, traumatic spinal cord injury or neuropathic pain.

In a particular embodiment of this aspect, the present invention provides for the use of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament useful for the treatment of Parkinson's disease and other movement disorders, Alzheimer's disease, or cognitive and negative symptoms in schizophrenia.

In one particular aspect, the present invention provides for the use of a compound of formula (I), as defined above, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament useful for the treatment of Parkinson's disease and other movement disorders.

The present invention also provides a method for the treatment and/or prevention of disorders for which the administration of D1 positive allosteric modulator is indicated, which comprises administering to a patient in need of such treatment an effective amount of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a method for the treatment and/or prevention of cognitive and negative symptoms in schizophrenia, cognitive impairment related to neuroleptic therapy, Mild Cognitive Impairment (MCI), impulsivity, Attention-Deficit Hyperactivity Disorder (ADHD), Parkinson's disease and other movement disorders, dystonia, Parkinson's dementia, Huntington's disease, dementia with Lewy Body, Alzheimer's disease, drug addiction, sleep disorders, apathy, traumatic spinal cord injury or neuropathic pain, which comprises administering to a patient in need of such treatment an effective amount of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof.

In a particular embodiment of this aspect, the present invention provides a method for the treatment of Parkinson's disease and other movement disorders, Alzheimer's disease, or cognitive and negative symptoms in schizophrenia, which comprises administering to a patient in need of such treatment of an effective amount of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof.

In one particular aspect, the present invention provides a method for the treatment of Parkinson's disease and other movement disorders, which comprises administering to a patient in need of such treatment of an effective amount of a compound of formula (I) as defined above, or a pharmaceutically acceptable salt thereof.

For use in medicine, the salts of the compound of formula (I) will be pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compound of formula (I) or of its pharmaceutically acceptable salts. Standard principles underlying the selection and preparation of pharmaceutically acceptable salts are described, for example, in Handbook of Pharmaceutical Salts: Properties, Selection and Use, ed. P. H. Stahl & C. G. Wermuth, Wiley-VCH, 2002. Suitable pharmaceutically acceptable salts of the compound of formula (I) include acid addition salts which may, for example, be formed by mixing a solution of the compound of formula (I) with a solution of a pharmaceutically acceptable acid.

It is to be understood that each individual atom present in formula (I), or in the formulae depicted hereinafter, may in fact be present in the form of any of its naturally occurring isotopes, with the most abundant isotope(s) being preferred. Thus, by way of example, each individual hydrogen atom present in formula (I), or in the formulae depicted hereinafter, may be present as a ¹H, ²H (deuterium) or ³H (tritium) atom, preferably ¹H. Similarly, by way of example, each individual carbon atom present in formula (I), or in the formulae depicted hereinafter, may be present as a ¹²C, ¹³C or ¹⁴C atom, preferably ¹²C.

The present invention includes within its scope solvates of the compounds of formula (I) above. Such solvates may be formed with common organic solvents or water.

The present invention also includes within its scope co-crystals of the compounds of formula (I) above. The technical term “co-crystal” is used to describe the situation where neutral molecular components are present within a crystalline compound in a definite stoichiometric ratio. The preparation of pharmaceutical co-crystals enables modifications to be made to the crystalline form of an active pharmaceutical ingredient, which in turn can alter its physicochemical properties without compromising its intended biological activity (see Pharmaceutical Salts and Co-crystals, ed. J. Wouters & L. Quere, RSC Publishing, 2012). Compounds according to the present invention may exist in different polymorphic forms. Although not explicitly indicated in the above formula, such forms are intended to be included within the scope of the present invention.

In a particular aspect according to the present invention, the compound of formula (I) is isolated in the form of a monohydrate as further described in the Examples.

The invention also includes within its scope pro-drug forms of the compounds of formula (I) and its various sub-scopes and sub-groups.

Activity in any of the above-mentioned therapeutic indications or disorders can of course be determined by carrying out suitable clinical trials in a manner known to a person skilled in the relevant art for the particular indication and/or in the design of clinical trials in general.

For treating diseases, compound of formula (I) or its pharmaceutically acceptable salts may be employed at an effective daily dosage and administered in the form of a pharmaceutical composition.

Therefore, the present invention also provides a pharmaceutical composition comprising the compound of formula (I) as depicted above, or a pharmaceutically acceptable salt thereof, in association with one or more pharmaceutically acceptable carriers.

To prepare a pharmaceutical composition according to the invention, one or more of the compounds of formula (I) or a pharmaceutically acceptable salt thereof is intimately admixed with a pharmaceutical diluent or carrier according to conventional pharmaceutical compounding techniques known to the skilled practitioner.

Suitable diluents and carriers may take a wide variety of forms depending on the desired route of administration, e.g., oral, rectal, parenteral or intranasal.

Pharmaceutical compositions according to the present invention can, for example, be administered orally, parenterally, i.e., intravenously, intramuscularly or subcutaneously, intrathecally, by inhalation or intranasally.

Pharmaceutical compositions suitable for oral administration can be solids or liquids and can, for example, be in the form of tablets, pills, dragees, gelatin capsules, solutions, syrups, chewing-gums and the like.

To this end the active ingredient may be mixed with an inert diluent or a non-toxic pharmaceutically acceptable carrier such as starch or lactose. Optionally, these pharmaceutical compositions can also contain a binder such as microcrystalline cellulose, gum tragacanth or gelatine, a disintegrant such as alginic acid, a lubricant such as magnesium stearate, a glidant such as colloidal silicon dioxide, a sweetener such as sucrose or saccharin, or colouring agents or a flavouring agent such as peppermint or methyl salicylate.

The invention also contemplates compositions which can release the active substance in a controlled manner. Pharmaceutical compositions which can be used for parenteral administration are in conventional form such as aqueous or oily solutions or suspensions generally contained in ampoules, disposable syringes, glass or plastics vials or infusion containers.

In addition to the active ingredient, these solutions or suspensions can optionally also contain a sterile diluent such as water for injection, a physiological saline solution, oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol, antioxidants such as ascorbic acid or sodium bisulphite, chelating agents such as ethylene diamine-tetra-acetic acid, buffers such as acetates, citrates or phosphates and agents for adjusting the osmolarity, such as sodium chloride or dextrose.

These pharmaceutical forms are prepared using methods which are routinely used by pharmacists.

The quantity of a compound of use in the invention required for the prophylaxis or treatment of a particular condition will vary depending on the compound chosen and the condition of the patient to be treated. In general, however, the daily dosage may range from 0.05 to 3000 mg, typically from 0.5 mg to 1000 mg for parenteral compositions.

The compound in accordance with the present invention, or a pharmaceutically acceptable salt thereof may be administered alone (monotherapy) or in combination with L-dopa (combination therapy). Alone or in combination with fractions of the L-dopa doses necessary to ameliorate motor disability in patients, the compounds of formula (I) according to the present invention, or pharmaceutical acceptable salts thereof, may be useful for the treatment of dyskinesia associated with administration of L-dopa. For example, if a compound of formula (I) in accordance with the present invention is used with fractions of the L-dopa doses given to patient or used alone to replace L-dopa, it is believed that compound of formula (I) according to the present invention will be effective against motor disability without inducing troublesome dyskinesia. Therefore it is believed that the compound according to the present invention may be useful for the treatment of motor deficits and levodopa-induced dyskinesia (LID).

Therefore, in one particular aspect, the present invention also provides a compound of formula (I), which is useful for the treatment of levodopa induced dyskinesia (LID).

Compound of formula (I) may be prepared by a process involving reacting an intermediate of formula (II) with an intermediate of formula (III),

Intermediate (III) may be conveniently reacted with intermediate of formula (II) in the presence of (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) or another coupling agent known to the person skilled in the art, in a suitable solvent, e.g. dimethylformamide, with an excess amount of a base, e.g. N,N-diisopropylethylamine.

Intermediate of formula (III) may be prepared by a process involving reaction of intermediates of formula (IV),

wherein

Z represents halogen or 1-hydroxy-1-methyl ethyl;

R^(a) represents tert-butyl dimethylsilyl; and

R^(c) represents hydrogen or tert-butoxycarbonyl.

In a first step, intermediate of formula (IV), wherein Z represents bromo, and R^(c) represents hydrogen, herein after referred to as intermediate (IVa), may be protected with an appropriate protective group, according to methods known to the skilled in the art to afford a compound of formula (IV), wherein Z represents bromo and R^(c) represents tert-butoxycarbonyl, herein after referred to as intermediate (IVb).

In a second step, a metal-halogen exchange reaction may be performed e.g. in the presence of n-BuLi, in a suitable solvent, e.g. tetrahydrofuran, at low temperature, in the presence of dry acetone under continuous flow, according to a method described in the accompanying Examples, to afford corresponding intermediate (IV) as described above wherein Z represents 1-hydroxy-1-methyl ethyl, herein after referred to as intermediate (IVc).

The tert-butoxycarbonyl (Boc) group (R^(c)) may then first be deprotected according to methods known to the person skilled in the art or as further described in the accompanying Examples, followed by deprotection of the trimethylsilyl group formed during Boc group deprotection and the tert-butyl dimethylsilyl group (R^(a)) to afford intermediate (III).

Intermediate of formula (IVa) may be prepared by a process involving reaction of an intermediate of formula (V), wherein Y is a halogen, e.g. bromo, and R^(a) is defined above for intermediate of formula (IV).

The reaction is conveniently effected in the presence of methyl magnesium chloride, in a suitable solvent e.g. tetrahydrofuran, at low temperature.

Intermediate (V) may be prepared by a 2 step process involving reaction of intermediate of formula (VI),

wherein Y is as defined above for intermediate of formula (V) and R^(a) represents hydrogen or tert-butyl-dimethylsilyl.

In a first step intermediate (VI) wherein R^(a) represents hydrogen is reacted with tert-butyldimethylsilyl chloride in the presence of a suitable base e.g. 4-dimethylamino-pyridine at room temperature, to afford intermediate (VI) wherein R^(a) represents tert-butyl-dimethylsilyl.

In a second step, intermediate (VI) wherein R^(a) represents tert-butyl-dimethylsilyl is reacted with N-Chlorosuccinimide (NCS), in a suitable solvent, e.g. THF to afford intermediate (V).

Intermediate (VI) wherein R^(a) represents hydrogen may be prepared by a process involving intermediate of formula (VII), wherein Y is as defined above for intermediate (V).

The reaction is conveniently effected in the presence of a strong base, e.g. sodium hydroxide, in a suitable solvent, e.g. mixture of ethanol and water, a high temperature.

Intermediate of formula (VII) may be prepared by a process involving reaction of intermediate (VIII),

wherein Y is as defined here above for intermediate of formula (V).

The reaction is conveniently effected in the presence of trimethylsilyltriflate and paraformaldehyde, in a suitable solvent e.g. dichloromethane.

Intermediate (VIII) may be prepared by a 2 steps process involving commercially available intermediate (IX),

wherein Y is as defined above for intermediate (V).

The reaction is conveniently effected according to the methods described in the accompanying examples or according to methods known to the person skilled in the art.

Intermediate of formula (II) may be prepared by a multi-step process involving reaction of intermediates of formula (X),

wherein

R¹ represents chloro, amino or nitro; and

R^(b) represents hydrogen or tert-butyl.

In a first step, intermediate of formula (X) wherein R¹ represents nitro and R^(b) represents tert-butyl, herein after referred to as intermediate (Xa), is reduced into the corresponding intermediate (X) wherein R¹ represents amino and R^(b) represents tert-butyl, herein after referred to as intermediate (Xb). The reaction is conveniently effected by Pd/C catalyzed hydrogenation under high pressure, in a suitable solvent e.g. methanol.

Intermediate (Xb) is transformed into corresponding intermediate (X) wherein R¹ represents chloro and R^(b) represents hydrogen, herein after referred to as intermediate (Xc), by adding concentrated hydrochloric acid and sodium nitrite, followed by further addition of hydrochloric acid and copper chloride (II). The reaction is conveniently effected at low temperature.

Intermediate (II) may then obtained directly from intermediate (Xc) by reaction with N-chlorosuccinimide according to the methods describe in the accompanying Examples or according to methods known to the person skilled in the art.

Intermediate of formula (Xa) may be prepared by a process involving an intermediate of formula (XI),

wherein R² represents hydrogen or methyl.

In a first step commercially available intermediate of formula (XI) wherein R² represents hydrogen, herein after referred to as intermediate (XIa), is reacted with methyl iodide in the presence of a strong base, e.g. potassium hydroxide, in a suitable solvent, e.g. dimethylformamide. The resulting intermediate (XI) wherein R² represents methyl, herein after referred to as intermediate (XIb) is then reacted with tert-butyl 2-chloroacetate in the presence of potassium tert-butoxide, in a suitable solvent, e.g. tetrahydrofuran, at low temperature, to afford intermediate (Xa).

Where a mixture of products is obtained from any of the processes described above for the preparation of compounds according to the invention, the desired product can be separated therefrom at an appropriate stage by conventional methods such as preparative HPLC; or column chromatography utilising, for example, silica and/or alumina in conjunction with an appropriate solvent system.

Where the above-described processes for the preparation of the compounds according to the invention give rise to mixtures of stereoisomers, these isomers may be separated by conventional techniques. In particular, where it is desired to obtain a particular enantiomer of a compound of formula (I) this may be produced from a corresponding mixture of enantiomers using any suitable conventional procedure for resolving enantiomers. Thus, for example, diastereomeric derivatives, e.g. salts, may be produced by reaction of a mixture of enantiomers of formula (I), e.g. a racemate, and an appropriate chiral compound, e.g. a chiral base. The diastereomers may then be separated by any convenient means, for example by crystallisation, and the desired enantiomer recovered, e.g. by treatment with an acid in the instance where the diastereomer is a salt. In another resolution process a racemate of formula (I) may be separated using chiral HPLC. Moreover, if desired, a particular enantiomer may be obtained by using an appropriate chiral intermediate in one of the processes described above. Alternatively, a particular enantiomer may be obtained by performing an enantiomer-specific enzymatic biotransformation, e.g. an ester hydrolysis using an esterase, and then purifying only the enantiomerically pure hydrolysed acid from the unreacted ester antipode. Chromatography, recrystallisation and other conventional separation procedures may also be used with intermediates or final products where it is desired to obtain a particular geometric isomer of the invention. Alternatively the non desired enantiomer may be racemized into the desired enantiomer, in the presence of an acid or a base, according to methods known to the person skilled in the art, or according to methods described in the accompanying Examples.

During any of the above synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W. Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 3rd edition, 1999. The protecting groups may be removed at any convenient subsequent stage utilising methods known from the art.

The compounds of formula (I) according to the present invention does not directly activate the dopamine D1 receptor, but potentiates the effect of D1 agonists or the endogenous ligand on D1 receptors, dopamine, through an allosteric mechanism, and is therefore D1 positive allosteric modulator (D1 PAM).

Dopamine and other D1 agonists directly activate the dopamine D1 receptor by themselves.

Assays have been designed to measure the effects of compounds in accordance with the present invention in the absence of dopamine (“activation assay”) and in the presence of dopamine (“potentiation assay”).

The activation assay measures the stimulation of the production of cyclic adenosinemonophosphate (cAMP) in the Homogeneous Time Resolved Fluorescent (HTRF) assay, with the maximum increase in cAMP by increasing concentrations of the endogenous agonist, dopamine, defined as 100% activation.

When tested, compounds of formula (I) according to the present invention lacks significant direct agonist-like effects in that it produces less than 20% of activation (compared to dopamine maximal response) when present in a concentration of 10 μM.

The potentiation assay measures the ability of compounds to increase the levels of cAMP produced by a low-threshold concentration of dopamine. The concentration of dopamine used ([EC20]) is designed to produce 20% stimulation compared to the maximal response (100%) seen with increasing the concentration of dopamine. To measure this potentiation increasing concentrations of the compound with the [EC20] of dopamine are incubated and the potentiation is measured as increases in cAMP production and concentration of compound which produces 50% of the potentiation of the cAMP levels is measured.

When tested in the cAMP HTRF assay, compound of formula (I) according to the the present invention has exhibited a value of pEC50 of greater than about 6.5 which shows that it is a D1 Positive Allosteric Modulator.

GABA_(A) receptor inhibition is known to be intimately linked to seizures and epilepsy. It is therefore desirable to develop compounds which are D1 Positive Allosteric Modulators and which at the same time minimize such effects.

When tested in a GABA-A receptor inhibition assay as described herein, it is therefore desirable that compound of formula (I) displays a percentage of inhibition of the GABA_(A) receptor of less than or equal to about 20%, ideally less than about 10%, appositely less than about 5%, when measured at a concentration of 10 μM of a compound of formula (I).

A problem which can be faced when developing compounds for use in therapy is the capacity for certain compounds to induce CYP450 enzymes. The induction of such enzymes may impact the exposure of such compounds or of other compounds which could be co-administered therewith to a patient, thereby potentially altering their respective safety or efficacy. It is therefore desirable to develop compounds which minimize such potential for induction.

The CYP450 induction potential of compound of formula (I) according to the present invention has been tested by measuring the potential increase of CYP450 activities in human hepatocytes treated with increasing concentrations of compounds according to the present invention.

When tested in the CYP3A4 induction assay according to the protocols described in the present patent application, it is desirable that compound of formula (I) according to the present invention exhibits a fold-induction value of at least about 2 times lower, ideally at least about 3 times lower, appositely at least about 4 times lower than the positive control compound (Rifampicin).

When developing a compound for use in therapy, it is important to have an idea of its elimination once it has been administrated into the body.

The clearance is a parameter which gives that information because it represents the volume of plasma (or blood) totally cleaned from the compound of interest by time unit. It is usually expressed as ml/min/kg or L/h. It can then be compared to any physiological blood flow (e.g. liver blood flow) to evaluate if the clearance is low, moderate or high.

When clearance is low, depending on the volume of distribution, one might expect to need a low dose and to get a relatively long duration of action. When clearance is high, again depending on the volume of distribution, one might expect to need a high dose and to get a relatively short duration of action.

The clearance is generally evaluated by using hepatocyte incubations and scaling calculations, assuming the main elimination pathway is metabolism, according to protocols described herein. The intrinsic clearance evaluated from hepatocytes is expressed in μl/min/10⁶ cells.

When tested in the clearance assay as described herein, the compound of formula (I) in accordance with the present invention advantageously exhibit a clearance of less than about 10 μl/min/10⁶ cells.

cAMP HTRF Assay

The particular conditions in which the compounds have been tested are described here below.

a. METHODS D1 Cell Culture

Cells were cultured at 37° C. in a humidified atmosphere of 5% CO₂. Cells were grown in DMEM-F12+GlutaMAX™-I medium (GIBCO®, Invitrogen, Merelbeke, Belgium) containing 10% fetal bovine serum (BioWhittaker®, Lonza, Verviers, Belgium), 400 μg/mL Geneticin (GIBCO®), 100 IU/mL Penicillin and 100 IU/mL Streptomycin (Pen-Strep solution, BioWhittaker®). LMtk (Ltk-) mouse fibroblast cells expressing the dopamine D1 receptor (BioSignal Inc, Montreal, Canada, now Perkin Elmer) were used as they have been shown to couple efficiently and give robust functional responses (Watts et al, 1995).

b. cAMP Assay

The measurement of changes in intracellular cyclic adenosinemonophopshpate (cAMP) was determined using the HTRF cAMP dynamic assay kit from CisBio (Codolet, France). Using homogenous time-resolved fluoresence technology, the assay is based on competition between native cAMP produced by cells and cAMP labelled with the dye d2. The tracer binding is determined by an anti-cAMP antibody labeled with cryptate. The effects of the compound alone (agonism) was determined by performing the assay in the absence of dopamine, whilst the effect of the compound as a positive allosteric modulator (PAM) was determined in the presence of an EC₂₀ concentration of dopamine. Cells (20,000 per well) are incubated in 384 plates for 1 hour at room temperature in a final volume of 20 pLHBSS (Lonza, with calcium, magnesium and HEPES buffer 20 mM, pH 7.4) containing: isobutyl methylxanthine (Sigma, 0.1 mM final), varying concentrations of test compound (typically 10^(−9.5)M to 10⁻⁴⁵M) in the presence and absence of dopamine (1.1 nM final). The reaction is then terminated and the cells lysed by adding the d2 detection reagent in lysis buffer (10 microL) and the cryptate reagent in lysis buffer (10 microl) according to manufacturer's instructions. This is then incubated for a further 60 min at room temperature and changes in HTRF fluorescent emission ratio determined according to manufacturer's instructions using an Envision plate reader (Perkin Elmer, Zaventem, Belgium) with laser excitation. All incubations were performed in duplicate and results were compared to a concentration-effect curve to dopamine. (10⁻¹¹M to 10⁻⁶M).

c. Data Analysis

Data was analyzed using Excel and PRISM (GraphPad Software) to obtain pEC₅₀ and Erel using the 4-parameter logistic equation (DeLean et al, 1978) where Erel is the fitted maximal response of the test compound minus basal expressed as a percentage relative to that obtained with dopamine which was defined as 100%.

The pEC₅₀ of a compound is the −log 10 of the concentration of the compound which produces 50% of the potentiation of the cAMP levels.

The Erel is the relative efficacy, defined as the maximal % potentiation produced by the compound compared to the maximal response produced by increasing concentrations of dopamine (Erel of 1=dopamine maximum response), has been measured.

When tested in the above assay, compound of formula (I) exhibits a value of pEC₅₀ of about 8.1 and an Erel value of about 68%.

Automated Patch Clamp Studies on the GABA_(A) Receptor Cells

CHO-K1 cells stably expressing human GABA_(A) receptor α1,β2 and γ2 subunits were used. The cells were harvested using trypsin and maintained in serum-free medium at room temperature. The cells were washed and re-suspended in extracellular solution before testing.

Patch Clamp Studies

Experiments on human GABA_(A) (α₁β₂γ₂) channels were conducted using an automated patch clamp assay (IonFlux™ HT). Compounds were tested at 3 concentrations (0.1, 1, and 10 μM) in 3 to 4 cells. The external solution for recording GABA_(A) currents was composed of sodium chloride 137 mM, potassium chloride 4 mM, calcium chloride 1.8 mM, magnesium chloride 1 mM, HEPES 10 mM, and glucose 10 mM. Both external and internal solutions were titrated with NaOH or KOH to obtain a pH of 7.35 or 7.3, respectively. The internal pipette solution contained potassium fluoride 70 mM, potassium chloride 60 mM, sodium chloride 70 mM, HEPES 5 mM, EGTA 5 mM, and Magnesium ATP 4 mM. The final concentration of vehicle used to dilute compounds was 0.33% DMSO in each well. Bicuculline (0.032 to 100 μM) was used as positive control inhibitor. GABA (15 μM) was used as agonist. All recordings were obtained from a holding potential of −60 mV.

The compound addition sequence was the following: one addition of the EC₈₀ concentration of GABA was added to establish baseline response. Each concentration of compound was applied for 30 seconds followed by the addition of 15 μM GABA in the presence of the compound for 2 seconds. The process was repeated with the next ascending concentration of compound. Peak inward currents in response to the GABA additions in the presence of a single concentration of compound were measured. All compound data have been normalized to the baseline peak current induced by addition of 15 μM GABA for 2 seconds.

When tested in the above mentioned assay, at a concentration of 10 μM, compound of represented by formula (I) exhibits a percentage of inhibition of the GABA_(A) receptor of less than about 0.1% measured at a concentration of 10 μM of a compound of formula (I).

Induction Assays

The following assays are aimed at determining the potential of the compound according to the present invention to induce CYP3A4 enzyme.

A. In Vitro Assessment of CYP3A4 Induction Potential Using Cryopreserved Human Hepatocytes

The objective of the human hepatocyte assay is to characterize the induction potential of Compound of formula (I) by measuring the CYP3A4 activities after 3-days of hepatocytes treatment with culture medium alone or culture medium containing increasing concentration of NCE.

To this aim, cryopreserved human hepatocytes from a single donor are seeded on a 48 well collagen coated plate so that the final seeding density is 0.2×10⁶ cells/well (final volume per well 0.25 mL). The cells are then incubated in seeding medium at 37° C., 95% humidity, 5% CO₂ to allow the cells to attach. After 4 h, the seeding medium is replaced with 0.25 mL of pre warmed serum free Williams E medium containing 100 IU/mL penicillin, 100 μg/mL streptomycin, 10 μg/mL insulin, 2 mM L glutamine and 0.1 μM hydrocortisone.

The next day, cells are dosed with test compound in assay medium at 1 μM (final DMSO concentration 0.1%). Positive control inducer, rifampicin (10 μM) is incubated alongside the test compound. Negative control wells are included where the test compound is replaced by vehicle solvent (0.1% DMSO in assay medium). Each test compound is dosed in triplicate. The cells are exposed to the culture medium for 72 h with medium changes every 24 hr.

For catalytic activity determination, solution of the CYP3A4 probe substrate, midazolam (final concentration 2.5 μM) is prepared in pre warmed assay medium. At the end of the 72 h exposure period, the media is replaced with the CYP3A4 probe substrate. The hepatocytes are incubated for 30 min. An aliquot is removed at the end of the incubation period and placed into an equal volume of methanol containing internal standard. The samples are centrifuged at 2500 rpm at 4° C. for 20 min. An aliquot of supernatant is diluted with deionised water and levels of 1-hydroxymidazolam is quantified using generic LC MS/MS methods.

The hepatocytes are solubilised in 0.1M sodium hydroxide at room temperature and protein content in each well determined using Pierce™ BCA Protein Assay Kit (Thermo Scientific) using bovine serum albumin as a standard.

Data Analysis

The fold-induction (increase in CYP activity) is obtained by taking the ratio of the measured enzymatic activity obtained in hepatocytes treated with Compound of formula (I), over the one obtained in control hepatocytes treated with vehicle. The fold-induction is calculated in the same way for positive control (Rifampicin). Then, the induction potential of the Compound of formula (I) is compared to that of rifampicin by calculating the ratio of the fold-induction seen with rifampicin over the fold-induction seen with Compound of formula (I).

When tested in the CYP3A4 induction assay at 1 μM concentration according to the protocols hereabove, compound of formula (I) according to the present invention exhibits a fold-induction value of at least about 7 times lower than the positive control compound (Rifampicin) at a concentration of 10 μM.

Using the same protocol described above, cells are dosed with test compound in assay medium at a range of concentrations from 0.03 to 10 μM (final DMSO concentration 0.1%). Positive control inducer, rifampicin is incubated alongside the test compound at a concentration range between 0.1 to 30 μM.

When comparing the highest fold-induction values coming from the protocol mentioned above, i.e. those obtained at the highest soluble concentration (3 μM) of compound of formula (I) according to the present invention and at 10 μM of the positive control (Rifampicin), compound of formula (I) exhibits an induction potential of about 4 times lower than that of the positive control compound (Rifampicin).

Azamulin Assay

Cryopreserved human hepatocytes (pool of 20 donors, BSU batch from Celsis/IVT/Bioreclamation) were thawed accordingly the provider's information. Viability (trypan blue exclusion) was higher than 75%. Pre-incubations (250 μL of hepatocytes suspension at 2×10⁶ hepatocytes/mL) were carried out with William's medium, containing 2 mM of glutamine and 15 mM of Hepes, in 48-well plates at +37° C., in an incubator (5% CO₂), under gentle agitation (vibrating agitator, Titramax 100, ca 300 rpm) during 30 min. After the pre-incubation, the incubation was initiated by adding to hepatocytes, 250 μL of culture medium (see composition above) containing UCB compound (1 μM) or midazolam (positive control) with or without azamulin (6 μM—specific CYP3A4/5 inhibitor). Final concentrations of UCB compound and azamulin in the incubates are 0.5 μM and 3 μM, respectively. The cell suspensions was rapidly re-homogenized by 2 in-out pipetting. After 0, 30, 60, 120, 180 and 240 minutes of incubation, reactions were stopped by transferring 50 μl of incubates into the appropriate well from 96-well plate containing 50 μL of ice cold acetonitrile with ketoconazole 1 μM as internal standard. Before each sampling, cell incubates are re-homogenized by 2 in out pipetting.

When incubated with human hepatocyte suspension at different concentrations the intrinsic clearance (Clint) of the compound of formula (I) according to the present invention is equal to 2.1±0.6 μl/min/10⁶ cells.

The following Examples illustrates the preparation of compounds of formula (I) according to the present invention.

EXAMPLES Abbreviations/Recurrent Reagents ACN: Acetonitrile

Brine: Saturated aqueous sodium chloride solution nBu: n-butyl tBu: tert-butyl

DCM: Dichloromethane DMAP: 4-Dimethylaminopyridine DMF: N,N-Dimethylformamide DMSO: Dimethylsulfoxide

EC_(20/50): concentration which produces 20%/50% of the maximum response Erel: relative efficacy

ES⁺: Electrospray Positive Ionisation Et: Ethyl EtOH: Ethanol

Et₂O: Diethyl ether EtOAc: Ethyl acetate

h: Hour HPLC: High Pressure Liquid Chromatography

HTRF: homogenous time-resolved fluorescence

LCMS: Liquid Chromatography Mass Spectrometry MeOH: Methanol

min.: minutes

NCS: N-Chlorosuccinimide

NMR: Nuclear magnetic resonance iPrOH: isopropanol rt: room temperature

SFC: Supercritical Fluid Chromatography TEA: Triethylamine THF: Tetrahydrofuran TLC: Thin Layer Chromatography

cAMP: cyclic adenosinemonophosphate

IUPAC names have been determined using Biovia Draw 16.1.

Analytical Methods

All reactions involving air or moisture-sensitive reagents were performed under a nitrogen or argon atmosphere using dried solvents and glassware. Commercial solvents and reagents were generally used without further purification, including anhydrous solvents when appropriate (generally Sure-Seal™ products from Aldrich Chemical Company or AcroSeal™ from ACROS Organics). In general reactions were followed by thin layer chromatography, HPLC or mass spectrometry analyses.

Crude materials could be purified by normal phase chromatography, (acidic or basic) reverse phase chromatography, chiral separation or recrystallization.

Products were generally dried under vacuum before final analyses and submission to biological testing.

All NMR spectra were obtained at 250 MHz, 300 MHz, 400 MHz or 500 MHz.

The compounds were studied in DMSO-d₆, CDCl₃ or MeOH-d₄ solution at a probe temperature of 300 K and at a concentration of 10 mg/mL. The instrument is locked on the deuterium signal of DMSO-d₆, CDCl₃ or CD₃OD. Chemical shifts are given in ppm downfield from TMS (tetramethylsilane) taken as internal standard.

1. Preparation of intermediate of formula (II)—2-(3,5-dichloro-1-methyl-indazol-4-yl)acetic Acid

1.1. Preparation of Intermediate (XIb)—1-Methyl-5-Nitro-Indazole

5-Nitro-1H-indazole (XIa) (3.00 kg, 18.4 mol) and DMF (30.0 L) are charged into a 50 L three-neck round-bottom flask at 15-30° C. KOH (2.06 Kg, 36.7 mol) is added in one portion into the reactor at 0-5° C. The mixture is stirred at 0-50° C. for 1 h. Methyl iodide (2.87 kg, 20.2 mol) is then added at 0-5° C. and the mixture is stirred for 3 h at 15-30° C. The reaction mixture is added into water (30 L) at 0-10° C. and the mixture is stirred for 10 min then filtered. The filter cake is washed with water (5 L) and dried. This overall procedure is carried out on 4 batches of the same size in parallel. The solids obtained from the four batches are combined to give 1-methyl-5-nitro-indazole (XIb) as a brown solid (10.0 kg, 42.3 mol, 75% purity (LC/MS), 57.5% yield) which is used in the next step without further purification.

¹H NMR (400 MHz, CDCl₃) δ 8.65 (s, 1H), 8.21 (d, J=9.17 Hz, 1H), 8.13 (s, 1H), 7.39 (d, J=9.17 Hz, 1H), 4.08 (s, 3H).

1.2. Preparation of Intermediate (Xa)—tert-butyl 2-(1-methyl-5-nitro-indazol-4-yl)acetate

t-BuOK (4.43 kg, 39.5 mol) and THF (30 L) are charged into a 50 L three-neck round-bottom flask and the mixture is cooled to −45/−35° C. under nitrogen and stirring. 1-Methyl-5-nitro-indazole (XIb) (3.50 kg, 19.7 mol) is then added in portions at −45/−35° C. Tert-butyl 2-chloroacetate (3.57 kg, 23.7 mol) is added dropwise at the same temperature and the mixture is stirred at 1 h. The mixture is warmed up to 15-30° C. and stirred for 5 h. The reaction is quenched by the addition of a saturated ammonium chloride solution (9 L) and water (2 L) is added. The organic layer is separated and the aqueous layer is extracted with ethyl acetate (2×5 L). The organic phases are combined, washed with brine (2 L), dried over Na₂SO₄, filtered and concentrated under vacuum. The crude product is purified by recrystallization with ethyl acetate (5 L). This overall procedure is carried out on 2 batches of the same size in parallel. The solids obtained from the two batches are combined and dried together to give tert-butyl 2-(1-methyl-5-nitro-indazol-4-yl)acetate as a yellow solid (Xa) (5.30 kg, 17.7 mol, 97.6% purity (LC/MS), 44.9% yield).

¹H NMR (400 MHz, CDCl₃) δ 8.18-8.20 (m, 2H), 7.37 (d, J=9.21 Hz, 1H), 4.27 (s, 2H), 4.14 (s, 3H), 1.44 (s, 9H).

1.3. Preparation of Intermediate (Xb)—tert-butyl 2-(5-amino-1-methyl-indazol-4-yl)acetate

Tert-butyl 2-(1-methyl-5-nitro-indazol-4-yl)acetate (Xa) (7.30 kg, 25.0 mol) and MeOH (76 L) are charged into a reactor. Argon is purged and Pd/C (50%, 760 g) is added. Hydrogen is added three times and the mixture is stirred at 50° C. under hydrogen atmosphere (50 psi) for 3 h. The reaction mixture is filtered and the solid is washed with MeOH (5 L). The mixture is concentrated to give tert-butyl 2-(5-amino-1-methyl-indazol-4-yl)acetate (Xb) as a brown oil (6.50 kg, 23.9 mol, 96.2% purity (LC/MS), 95.4% yield) which is used in the next step without further purification.

¹H NMR (400 MHz, CDCl₃) δ 7.72 (s, 1H), 7.27 (d, J=8.80 Hz, 1H), 6.91 (d, J=8.80 Hz, 1H), 4.60 (s, 2H), 3.93 (s, 3H), 3.68 (s, 2H), 1.38 (s, 9H).

1.4. Preparation of Intermediate (Xc)—2-(5-chloro-1-methyl-indazol-4-yl)acetic Acid

Tert-butyl 2-(5-amino-1-methyl-indazol-4-yl)acetate (Xb) (2.00 kg, 7.65 mol) and concentrated HCl (10.0 L, 12M) are charged into a 50 L three-neck round bottom flask and the mixture is cooled to −10/−5° C. and stirred. A water solution (5 L) of sodium nitrite (686 g, 9.95 mol) is added dropwise at −10/−5° C. and stirred for 30 min. CuCl (833 g, 8.42 mol) and concentrated HCl (10.0 L, 12M) are charged into a 20 L three-neck round bottom flask and the mixture is stirred for 30 min. at −10/−5° C., then added into the other reactor. The mixture is stirred at −10/−5° C. for 1 h, then at 10-30° C. for 16 h. The reaction mixture is filtered and the solid washed with water. This overall procedure is carried out on 3 batches of the same size in parallel. The solids obtained from the three batches are combined and dried together to give 2-(5-chloro-1-methyl-indazol-4-yl)acetic acid (Xc) as a yellow solid (4.00 kg, 16.3 mol, 92% purity (LC/MS), 71.3% yield) which is used in the next step without further purification.

1.5. Preparation of 2-(3,5-dichloro-1-methyl-indazol-4-yl)acetic Acid (II)

2-(5-Chloro-1-methyl-indazol-4-yl)acetic acid (Xc) (1.30 kg, 5.79 mol) and DMF (6.5 L) are charged into a 50 L three-neck round bottom flask at 20° C. N-Chlorosuccinimide (772 g, 5.79 mol) is added portionwise at 20° C. and the mixture is stirred at 20° C. for 2 h. The reaction mixture is poured into water (25 L) and filtered. The crude product is triturated with isopropyl ether:ethyl acetate (3:1) (7.0 L) at 20° C. for 2 h then filtered and dried. This overall procedure is carried out on 3 batches of the same size in parallel. The solids obtained from the three batches are combined to give 2-(3,5-dichloro-1-methyl-indazol-4-yl)acetic acid (II) (2.1 kg, 7.9 mol, 97.5% purity (LC/MS), 46% yield).

¹H NMR (400 MHz, CDCl₃) δ 12.67 (s, 1H), 7.68 (d, J=9.05 Hz, 1H), 7.53 (d, J=9.05 Hz, 1H), 4.20 (s, 2H), 4.02 (s, 3H).

2. Preparation of Compound of Formula (I) 2-(3,5-dichloro-1-methyl-indazol-4-yl)-1-[(1S,3R)-3-(hydroxymethyl)-5-(1-hydroxy-1-methyl-ethyl)-1-methyl-3,4-dihydro-1H-isoquinolin-2-yl]ethanone

2.1. Preparation of Intermediate (IX) (2R)-2-amino-3-(2-bromophenyl)propan-1-ol—a6

(2R)-2-amino-3-(2-bromophenyl)propanoic acid a5 (34.0 kg, 139 mol) and THF (238 L) are charged into a reactor. Sodium borohydride (15.6 kg, 413 mol) is added slowly at 20-30° C. A solution of iodine (35.3 kg, 139 mol) in dry THF (20.0 L) is added slowly at 0-10° C. and the reaction mixture is stirred at 70° C. for 12 h. The reaction was quenched with methanol (70.0 L) at 0° C. and heated to 80° C. for 30 min. The mixture was cooled down, concentrated under vacuum and the residue was suspended in NaOH (30.0 L, 2N), then filtered. The filter cake was dried under vacuum to give (2R)-2-amino-3-(2-bromophenyl)propan-1-ol a6 as a white solid (31.0 kg, 135 mol, 96.7% yield) which is used in the next step without further purification.

¹H NMR (400 MHz, CDCl₃) δ 7.57 (d, J=7.7 Hz, 1H), 7.21-7.29 (m, 2H), 7.07-7.15 (m, 1H), 3.66 (dd, J=10.5, 3.6 Hz, 1H), 3.41 (dd, J=10.5, 7.2 Hz, 1H), 3.18-3.29 (m, 1H), 2.95 (dd, J=13.5, 5.5 Hz, 1H), 2.70 (dd, J=13.5, 8.2 Hz, 1H), 1.51-1.91 (m, 3H).

2.2. Preparation of Intermediate of Formula (VIII) (4R)-4-[(2-bromophenyl)methyl]oxazolidin-2-one—a7

(2R)-2-amino-3-(2-bromophenyl)propan-1-ol a6 (31.0 kg, 135 mol) and dichloromethane (220 L) are charged into a reactor. Triphosgene (13.9 kg, 47.1 mol) is added at room temperature then N,N-diisopropylethylamine (39.1 kg, 303 mol) is slowly added at 0-10° C. The reaction mixture is stirred at 0-10° C. for 1 h then washed with water (50.0 L) twice, dried with anhydrous sodium sulfate and filtered to give (4R)-4-[(2-bromophenyl)methyl]oxazolidin-2-one a7 as a solution in dichloromethane which is used directly in the next step.

2.3. Preparation of Intermediate (VII) (10aR)-9-bromo-1,5,10,10a-tetrahydrooxazolo[3,4-b]isoquinolin-3-one a8

A solution of (4R)-4-[(2-bromophenyl)methyl]oxazolidin-2-one a7 (135 mol) in dichloromethane (220 L) is charged into a reactor and cooled down to 0-5° C. Trimethylsilyl triflate (35.9 kg, 162 mol) and paraformaldehyde (13.3 kg, 148 mol) are added at 0-5° C., then stirred for 2 h at 15-20° C. Water (170 L) is added into the mixture which is then extracted twice with dichloromethane (50.0 L). the organic layer is dried with anhydrous sodium sulfate, filtered and concentrated under vacuum. A mixture of petroleum ether:ethyl acetate (1:1, 45.0 L) is added and the mixture is stirred at room temperature for 6 h and filtered. The solid was dried to get (10aR)-9-bromo-1,5,10,10a-tetrahydrooxazolo[3,4-b]isoquinolin-3-one a8 as an off-white solid (29.0 kg, 80.2% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.45-7.52 (m, 1H), 7.08-7.14 (m, 2H), 4.83 (d, J=17.0 Hz, 1H), 4.62 (t, J=8.4 Hz, 1H), 4.36 (d, J=17.0 Hz, 1H), 4.21 (dd, J=8.6, 4.9 Hz, 1H), 3.91-3.99 (m, 1H), 3.25 (dd, J=16.3, 4.2 Hz, 1H), 2.67 (dd, J=16.1, 11.0 Hz, 1H).

2.4. Preparation of Intermediates (VI) 2.4.1. [(3R)-5-bromo-1,2,3,4-tetrahydroisoquinolin-3-yl]methanol a9

Ethanol (120 L) and water (60.0 L) are mixed into a reactor. (10aR)-9-bromo-1,5,10,10a-tetrahydrooxazolo[3,4-b]isoquinolin-3-one a8 (29.7 kg, 111 mol) is added then sodium hydroxide (13.3 kg, 332 mol) is slowly added at 15-20° C. The reaction mixture is stirred at 90° C. for 2 h then cooled down to room temperature. Water (300 L) is added into the mixture which is centrifugated. The centrifugal cake is dried in circulation oven to give [(3R)-5-bromo-1,2,3,4-tetrahydroisoquinolin-3-yl]methanol a9 as a white solid (23.7 kg, 88.3% yield) which is used in the next step without further purification.

¹H NMR (400 MHz, CDCl₃) δ 7.37-7.47 (m, 1H), 6.95-7.08 (m, 2H), 4.00-4.10 (m, 2H), 3.85 (dd, J=10.9, 3.7 Hz, 1H), 3.57 (dd, J=10.9, 7.9 Hz, 1H), 3.06 (ddt, J=11.3, 7.6, 4.1, 4.1 Hz, 1H), 2.79 (dd, J=17.1, 4.4 Hz, 1H), 2.40 (dd, J=17.1, 10.9 Hz, 1H), 1.93 (br s, 2H).

2.4.2. [(3R)-5-bromo-1,2,3,4-tetrahydroisoquinolin-3-yl]methoxy-tert-butyl-dimethyl-silane a10

[(3R)-5-bromo-1,2,3,4-tetrahydroisoquinolin-3-yl]methanol a9 (23.7 kg, 97.8 mol) and dichloromethane (240 L) are charged into a reactor. DMAP (120 g, 0.98 mol) and imidazole (13.3 kg, 196 mol) are added. Tert-butyldimethylsilyl chloride (TBSCl) (17.7 kg, 117 mol) is slowly added at 15-20° C. and the mixture is stirred for 12 h. Ammonium chloride (100 L) is added into the mixture. The organic phase was separated, washed with water (50.0 L), dried with anhydrous sodium sulfate, filtered and concentrated under vacuum to give [(3R)-5-bromo-1,2,3,4-tetrahydroisoquinolin-3-yl]methoxy-tert-butyl-dimethyl-silane a10 as a yellow oil (37.6 kg, 86% purity, 93% yield) which is used in the next step without further purification.

¹H NMR (400 MHz, CDCl₃) δ 7.36-7.45 (m, 1H), 7.01 (d, J=4.6 Hz, 1H), 4.01-4.13 (m, 2H), 3.84 (dd, J=9.9, 3.7 Hz, 1H), 3.64 (dd, J=9.8, 7.2 Hz, 1H), 2.96-3.08 (m, 1H), 2.75 (dd, J=17.0, 4.2 Hz, 1H), 2.44 (dd, J=17.0, 10.8 Hz, 1H), 1.76-2.20 (m, 2H), 0.89-0.97 (m, 9H), 0.08-0.14 (m, 6H).

2.5. Preparation of Intermediate (V) [(3R)-5-bromo-3,4-dihydroisoquinolin-3-yl]methoxy-tert-butyl-dimethyl-silane a11

[(3R)-5-bromo-1,2,3,4-tetrahydroisoquinolin-3-yl]methoxy-tert-butyl-dimethyl-silane al 0 (3.42 kg, 8.31 mol) and THF (30.0 L) are charged into a reactor. N-Chlorosuccinimide (NCS) (1.17 kg, 8.73 mol) is slowly added at room temperature and the mixture is stirred at 25° C. for 30 min. A solution of KOH (1.52 kg, 27.1 mol) in dry methanol (7.00 L) is slowly added at room temperature and the reaction is stirred at 25° C. for 1 h. The reaction is quenched with water (10.0 L) and extracted with petroleum ether:ethyl acetate (1:2, 5.00 L). The organic layer is separated, washed with brine (10.0 L), dried with anhydrous sodium sulfate and filtered. This overall procedure is carried out on 10 batches of the same size in parallel and the 10 reaction filtrates are combined and concentrated under vacuum to give [(3R)-5-bromo-3,4-dihydroisoquinolin-3-yl]methoxy-tert-butyl-dimethyl-silane a11 as a brown oil (28.0 kg, crude) which is used in the next step without further purification.

¹H NMR (400 MHz, CDCl₃) δ 8.24 (d, J=2.6 Hz, 1H), 7.58 (dd, J=7.8, 1.2 Hz, 1H), 7.12-7.25 (m, 2H), 4.03 (dd, J=9.5, 4.0 Hz, 1H), 3.67-3.77 (m, 2H), 3.07 (dd, J=17.0, 6.2 Hz, 1H), 2.68 (dd, J=17.1, 10.9 Hz, 1H), 0.88-0.91 (m, 9H), 0.07 (d, J=1.5 Hz, 6H).

2.6. Preparation of Intermediates of Formula (IV) 2.6.1. [(1S,3R)-5-bromo-1-methyl-1,2,3,4-tetrahydroisoquinolin-3-yl]methoxy-tert-butyl-dimethyl-silane (IVa)

[(3R)-5-bromo-3,4-dihydroisoquinolin-3-yl]methoxy-tert-butyl-dimethyl-silane a11 (3.10 kg, 8.75 mol) and THF (20.0 L) are charged into a reactor. The mixture is cooled down to 0° C. and methylmagnesium chloride (3M, 11.6 L) is added. The mixture is stirred at 20° C. for 12 h. The reaction is quenched with a saturated solution of ammonium chloride. The phases are separated and the aqueous layer is extracted twice with petroleum ether:ethyl acetate (3:1, 5.00 L). The combined organic phases are washed with brine (10.0 L), dried over anhydrous sodium sulfate and filtered. This overall procedure is carried out on 9 batches of the same size in parallel and the nine reaction filtrates are combined and concentrated under vacuum.

The crude mixture is purified by silica gel chromatography with petroleum ether:ethyl acetate (10:1) to give [(1S,3R)-5-bromo-1-methyl-1,2,3,4-tetrahydroisoquinolin-3-yl]methoxy-tert-butyl-dimethyl-silane (IVa) as a brown oil (4.60 kg, 99.7% purity, 15.7% yield).

¹H NMR (400 MHz, DMSO-d₆) δ 7.41 (dd, J=7.7, 0.9 Hz, 1H), 7.12-7.18 (m, 1H), 7.03-7.11 (m, 1H), 4.12 (q, J=6.8 Hz, 1H), 3.62 (d, J=5.7 Hz, 2H), 3.07-3.17 (m, 1H), 2.67-2.76 (m, 1H), 2.26 (dd, J=16.9, 10.0 Hz, 1H), 2.12 (br s, 1H), 1.32 (d, J=6.8 Hz, 3H), 0.84-0.93 (m, 9H), 0.07 (d, J=0.9 Hz, 6H).

2.6.2. tert-butyl (1S,3R)-5-bromo-3-[[tert-butyl(dimethyl)silyl]oxymethyl]-1-methyl-3,4-dihydro-1H-isoquinoline-2-carboxylate (IVb)

[(1S,3R)-5-bromo-1-methyl-1,2,3,4-tetrahydroisoquinolin-3-yl]methoxy-tert-butyl-dimethyl-silane (IVa) (1.85 kg, 4.99 mol) and dichloromethane (13.0 L) are charged in a reactor. N,N-diisopropylethylamine (1.94 kg, 14.9 mol) and di-tert-butyl dicarbonate (1.14 kg, 5.24 mol) are added at room temperature and the mixture is stirred for 12 h. The reaction mixture is washed twice with a saturated ammonium chloride solution (10.0 L), the organic layer is dried with anhydrous sodium sulfate and filtered. This overall procedure is carried out on 2 batches of the same size in parallel and the two reaction filtrates are combined and concentrated under vacuum. The crude mixture is purified by silica gel chromatography with petroleum ether:ethyl acetate (30:1) to give tert-butyl (1S,3R)-5-bromo-3-[[tert-butyl(dimethyl)silyl]oxymethyl]-1-methyl-3,4-dihydro-1H-isoquinoline-2-carboxylate (IVb) as a yellow oil (4.00 kg, 99.5% purity, 85.2% yield).

¹H NMR (400 MHz, DMSO-d₆) δ 7.50 (d, J=7.9 Hz, 1H), 7.22 (br d, J=6.7 Hz, 1H), 7.06-7.18 (m, 1H), 4.84 (br s, 1H), 4.12 (br s, 1H), 3.46 (br d, J=15.4 Hz, 2H), 2.94 (br dd, J=15.8, 5.2 Hz, 1H), 2.71 (br t, J=9.5 Hz, 1H), 1.45 (s, 9H), 1.28 (br s, 3H), 0.81 (s, 9H), −0.08 (s, 6H).

2.6.3. tert-butyl (1S,3R)-3-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-(1-hydroxy-1-methyl-ethyl)-1-methyl-3,4-dihydro-1H-isoquinoline-2-carboxylate (IVc)

A solution of tert-butyl (1S,3R)-5-bromo-3-[[tert-butyl(dimethyl)silyl]oxymethyl]-1-methyl-3,4-dihydro-1H-isoquinoline-2-carboxylate (IVb) (42.5 g, 90.3 mmol) in dry THF (0.5 M solution) and a commercial solution of n-Buthylithium in Hexanes (1.6 M solution) were pumped at respectively 6.0 ml/min (1.0 equiv) and 2.46 mL/min (1.3 equiv.) and were mixed in a glass microchip cooled at −40° C. The mixed flow stream was pumped through the reaction zone 1 of the microchip (0.3 mL) and was then combined with a solution of dry acetone (13.5 M) pumped at 6.0 mL/min (27 equiv.). The resulting stream was then passed through the reaction zone 2 of the microchip (0.7 mL) at −40° C. Finally, the global flow stream exiting the reactor was collected and quenched at room temperature in a saturated solution of aqueous ammonium chloride. When all the feed solutions were consumed, a bilayer reaction mixture was obtained. The aqueous layer was separated from the organic layer, and then extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate and concentrated under vacuum. A yellow oil was obtained (46.5 g) and was purified by SFC chromatography on a GreenSep Nitro column (10p, 5×22.3 using CO₂ 98%/EtOH 2% eluent). The solvent was removed under vacuum to yield to a white solid, tert-butyl (1S, 3R)-3-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-(1-hydroxy-1-methyl-ethyl)-1-methyl-3,4-dihydro-1H-isoquinoline-2-carboxylate (IVc) (25 g, 56 mmol, 62% yield).

UPLC_MS basic 1 pic @ 3.83 min (ES+): 350 (M-Boc+H)⁺, 332 (M−Boc−H₂O+H)+, 100% purity.

¹H NMR (400 MHz, DMSO-d₆) δ 7.44 (d, J=7.9 Hz, 1H), 7.19 (dt, J=8.1, 5.2 Hz, 1H), 7.09 (t, J=9.0 Hz, 1H), 4.99 (s, 1H), 4.87 (dq, J=13.4, 6.4 Hz, 1H), 4.11 (s, 1H), 3.96 (t, J=14.9 Hz, 1H), 3.48 (dd, J=9.4, 4.1 Hz, 1H), 2.98 (dd, J=16.5, 5.0 Hz, 1H), 2.89 (t, J=9.6 Hz, 1H), 1.65 (s, 3H), 1.58 (s, 3H), 1.55 (d, J=2.5 Hz, 9H), 1.34 (dd, J=20.5, 6.6 Hz, 3H), 0.90 (s, 9H), 0.08 (d, J=7.2 Hz, 3H), −0.00 (s, 3H).

2.7. Preparation of intermediate (III) 2-[(1S,3R)-3-(hydroxymethyl)-1-methyl-1,2,3,4-tetrahydroisoquinolin-2-ium-5-yl]propan-2-ol Chloride 2.7.1. tert-butyl-dimethyl-[[(1S,3R)-1-methyl-5-(1-methyl-1-trimethylsilyloxy-ethyl)-1,2,3,4-tetrahydroisoquinolin-3-yl]methoxy]silane—a15

Tert-butyl (1S,3R)-3-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-(1-hydroxy-1-methyl-ethyl)-1-methyl-3,4-dihydro-1H-isoquinoline-2-carboxylate (IVc) (148 g, 87% purity, 287 mmol) is dissolved in 1000 mL dichloromethane and transferred to a 2 liter double walled reactor. 2,6-Lutidine (100 mL, 860 mmol) is added and the jacket temperature is set at −2° C. Trimethylsilyl trifluoromethanesulfonate (154 g, 129 mL, 692 mmol) is added over 40 min via an addition funnel. Two hours after the start of addition, the reaction is quenched by adding 650 mL of an aqueous citric acid solution (1M) and the temperature of the mixture is brought back to 20° C. One hour after the start of the quench, the layers are separated. The organic layer is washed twice with 350 mL of an aqueous solution of citric acid (1M). The organic layer is stirred with 750 mL of aqueous sodium carbonate (10% w/w) for 10 min before separation of the layers. The organic layer is dried over anhydrous sodium sulfate. The organic layer is then filtered and the filtrate is concentrated under vacuum at 40° C. providing a yellow oil (128 g) of tert-butyl-dimethyl-[[(1S,3R)-1-methyl-5-(1-methyl-1-trimethylsilyloxy-ethyl)-1,2,3,4-tetrahydroisoquinolin-3-yl]methoxy]silane a15 which is used in the next step without further purification.

¹H NMR (400 MHz, CDCl₃) δ 7.19 (d, J=7.7 Hz, 1H), 7.07 (t, J=7.7 Hz, 1H), 7.00 (d, J=7.6 Hz, 1H), 4.24 (q, J=6.8 Hz, 1H), 3.75 (dd, J=9.7, 4.4 Hz, 1H), 3.60 (dd, J=9.7, 7.0 Hz, 1H), 3.54 (dd, J=16.3, 3.5 Hz, 1H), 3.15 (ddt, J=10.9, 7.4, 4.0 Hz, 1H), 2.52 (dd, J=16.3, 10.9 Hz, 1H), 1.66 (d, J=14.6 Hz, 6H), 1.52-1.43 (m, 3H), 0.92 (q, J=1.2 Hz, 9H), 0.14 (q, J=1.2 Hz, 2H), 0.09 (d, J=1.1 Hz, 6H), 0.00 (q, J=1.2, 0.8 Hz, 9H).

2.7.2. 2-[(1S,3R)-3-(hydroxymethyl)-1-methyl-1,2,3,4-tetrahydroisoquinolin-2-ium-5-yl]propan-2-ol Chloride Intermediate (III)

In a three-neck round bottom flask equipped with a mechanical stirrer, tert-butyl-dimethyl-[[(1S,3R)-1-methyl-5-(1-methyl-1-trimethylsilyloxy-ethyl)-1,2,3,4-tetrahydroisoquinolin-3-yl]methoxy]silane a15 (20.0 g, 47.4 mmol) is dissolved in 220 mL of isopropanol. To this solution, 42.3 mL of hydrochloric acid in iso-propanol (5-6 M, around 5 eq.) are added. 45 min after addition of hydrochloric acid, a 100 mg of seeds of the desired product are introduced. After 7 hours at room temperature, the reaction mixture is filtered over a sintered glass filter. The filtercake is washed with 40 mL isopropanol and dried under vacuum at room temperature overnight. 11.1 g of 2-[(1S,3R)-3-(hydroxymethyl)-1-methyl-1,2,3,4-tetrahydroisoquinolin-2-ium-5-yl]propan-2-ol chloride (III) are obtained as a pinkish solid. The yield over the two deprotection steps is 91%.

¹H NMR (400 MHz, CD₃OD) δ 7.46 (dd, J=7.8, 1.3 Hz, 1H), 7.28 (t, J=7.8 Hz, 1H), 7.21 (dd, J=7.8, 1.3 Hz, 1H), 4.63 (q, J=6.9 Hz, 1H), 3.97 (dd, J=11.7, 3.8 Hz, 1H), 3.88 (dd, J=17.2, 4.3 Hz, 1H), 3.78 (dd, J=11.8, 6.1 Hz, 1H), 3.66-3.56 (m, 1H), 3.14 (dd, J=17.2, 11.4 Hz, 1H), 1.73 (d, J=6.8 Hz, 3H), 1.64 (d, J=4.8 Hz, 6H). OH and NH protons are not observed.

2.8. Preparation of Compound of Formula (I) 2-(3,5-dichloro-1-methyl-indazol-4-yl)-1-[(1S,3R)-3-(hydroxymethyl)-5-(1-hydroxy-1-methyl-ethyl)-1-methyl-3,4-dihydro-1H-isoquinolin-2-yl]ethenone

In a 100 mL Easymax reactor equipped with a mechanical stirrer, 2-(3,5-dichloro-1-methyl-indazol-4-yl)acetic acid (II) (4.00 g, 15.4 mmol), 2-[(1S,3R)-3-(hydroxymethyl)-1-methyl-1,2,3,4-tetrahydroisoquinolin-2-ium-5-yl]propan-2-ol chloride (III) (4.46 g, 16.4 mmol) and 48 mL of DMF are charged. The suspension is stirred at 20° C. and then cooled by setting the jacket temperature to −2° C. Once the temperature of the mixture is below 3° C., N,N-diisopropylethylamine (9.5 mL, 54 mmol) is added. (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (6.4 g, 17 mmol) is added in four portions over 1 hour. The mixture is stirred for 1 h 45 before setting the jacket temperature at 15° C. 16 mL of water are then added over the course of a few minutes. 15 min later, 30 mg of solid product are added as seeds to initiate the crystallization. The jacket temperature is set at 20° C. Half an hour later, 16 mL of water are added over 17 min. Stirring of the suspension is pursued for 2 h 15 at 20° C. before being filtered on sintered glass. The filtercake is washed with two portions of 20 mL of water and then dried at 50° C. overnight under vacuum yielding 6.03 g of 2-(3,5-dichloro-1-methyl-indazol-4-yl)-1-[(1S,3R)-3-(hydroxymethyl)-5-(1-hydroxy-1-methyl-ethyl)-1-methyl-3,4-dihydro-1H-isoquinolin-2-yl]ethenone (I) (crude material).

A recristallization is carried out on 5.00 g of the crude material obtained by first suspending in 50 mL acetonitrile. The jacket temperature is set to 70° C. Once the solid has dissolved and the mass temperature has reached 66° C., 720 μl of water are added. The mass temperature is then cooled to 59° C. and 125 mg of solid product is added as seeding material. The mass temperature is then decreased to 55° C. over 25 min at which stage crystallization is occurring. The jacket temperature is then decreased over two hours from 58° C. down to 20° C. After 50 min, the suspension is filtered and the filtercake is washed with 7.5 mL acetonitrile. The filtercake is then dried under vacuum at 45° C. overnight and 2 hours at 50° C. providing 4.04 g of 2-(3,5-dichloro-1-methyl-indazol-4-yl)-1-[(1S,3R)-3-(hydroxymethyl)-5-(1-hydroxy-1-methyl-ethyl)-1-methyl-3,4-dihydro-1H-isoquinolin-2-yl]ethenone (I) as an off-white powder (hydrate form) Yield=64%.

¹H NMR (400 MHz, DMSO-d₆) δ 7.65 (dd, J=9.0, 2.2 Hz, 1H), 7.52 (dd, J=9.0, 2.1 Hz, 1H), 7.37 (ddd, J=19.6, 7.6, 1.7 Hz, 1H), 7.25-7.03 (m, 2H), 5.30 (q, J=6.5 Hz, 0.3H), 5.16-4.99 (m, 1.7H), 4.99-4.84 (m, 0.7H), 4.63-4.30 (m, 3.3H), 4.17-3.93 (m, 4H), 3.28 (dt, J=10.5, 5.1 Hz, 1.3H), 3.10-2.85 (m, 1.7H), 1.56 (dd, J=13.2, 6.9 Hz, 6.7H), 1.24 (d, J=6.5 Hz, 2.3H). 

1. A compound which is 2-(3,5-dichloro-1-methyl-indazol-4-yl)-1-[(1S,3R)-3-(hydroxymethyl)-5-(1-hydroxy-1-methyl-ethyl)-1-methyl-3,4-dihydro-1H-isoquinolin-2-yl]ethanone of formula (I), or a pharmaceutically acceptable salt thereof,


2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled)
 6. A pharmaceutical composition comprising a compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier.
 7. A method for the treatment and/or prevention of disorders for which the administration of D1 positive allosteric modulator is indicated, which comprises administering to a patient in need of such treatment an effective amount of a compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof.
 8. A method for the treatment and/or prevention of cognitive and negative symptoms in schizophrenia, cognitive impairment related to neuroleptic therapy, Mild Cognitive impairment (MCI), impulsivity, Attention-Deficit Hyperactivity Disorder (ADHD), Parkinson's disease and other movement disorders, dystonia, Parkinson's dementia, Huntington's disease, dementia with Lewy Body, Alzheimer's disease drug addiction, sleep disorders, apathy, traumatic spinal cord injury or neuropathic pain, which comprises administering to a patient in need of such treatment an effective amount of a compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof.
 9. A method for the treatment of Parkinson's disease and other movement disorders, Alzheimer's disease, or cognitive and negative symptoms in schizophrenia which comprises administering to a patient in need of such treatment of an effective amount of a compound of formula (I) as defined in claim 1, or a pharmaceutically acceptable salt thereof. 